Development of a Novel Chikungunya Virus-Like Replicon Particle for Rapid Quantification and Screening of Neutralizing Antibodies and Antivirals

ABSTRACT Chikungunya fever is a mosquito-transmitted infectious disease that induces rash, myalgia, and persistent incapacitating arthralgia. At present, no vaccines or antiviral therapies specific to Chikungunya virus (CHIKV) infection have been approved, and research is currently restricted to biosafety level 3 containment. CHIKV-like replicon particles (VRPs) are single-cycle infectious particles containing viral structure proteins, as well as a defective genome to provide a safe surrogate for living CHIKV to facilitate the testing of vaccines and antivirals. However, inefficient RNA transfection and the potential emergence of the competent virus through recombination in mammalian cells limit VRP usability. This study describes a transfection-free system for the safe packaging of CHIK VRP with all necessary components via transduction of mosquito cell lines using a single baculovirus vector. We observed the release of substantial quantities of mosquito cell-derived CHIK VRP (mos-CHIK VRP) from baculovirus-transduced mosquito cell lines. The VRPs were shown to recapitulate viral replication and subgenomic dual reporter expression (enhanced green fluorescent protein [eGFP] and luciferase) in infected host cells. Interestingly, the rapid expression kinetics of the VRP-expressing luciferase reporter (6 h) makes it possible to use mos-CHIK VRPs for the rapid quantification of VRP infection. Treatment with antivirals (suramin or 6-azauridine) or neutralizing antibodies (monoclonal antibodies [MAbs] or patient sera) was shown to inhibit mos-CHIK VRP infection in a dose-dependent manner. Ease of manufacture, safety, scalability, and high throughput make mos-CHIK VRPs a highly valuable vehicle for the study of CHIKV biology, the detection of neutralizing (NT) antibody activity, and the screening of antivirals against CHIKV. IMPORTANCE This study proposes a transfection-free system that enables the safe packaging of CHIK VRPs with all necessary components via baculovirus transduction. Those mosquito cell-derived CHIK VRP (mos-CHIK VRPs) were shown to recapitulate viral replication and subgenomic dual reporter (enhanced green fluorescent protein [eGFP] and luciferase) expression in infected host cells. Rapid expression kinetics of the VRP-expressing luciferase reporter (within hours) opens the door to using mos-CHIK VRPs for the rapid quantification of neutralizing antibody and antiviral activity against CHIKV. To the best of our knowledge, this is the first study to report a mosquito cell-derived alphavirus VRP system. Note that this system could also be applied to other arboviruses to model the earliest event in arboviral infection in vertebrates.

perform several validation studies for their VRP system and report that the VRPs recapitulate authentic CHIK life cycle in several assays including in vitro neutralization with human patient serum, inhibition of replication with small molecule inhibitors, etc. Finally, in the viral neutralization assays, this VRP system showed higher sensitivity and reproducibility than lentivirus-based CHIKV pseudoparticles. These results demonstrate that this newly developed mos-VRP-CHIKV can serve as a better tool to study CHIKV under BSL2 conditions. Overall, this manuscript describes the development of a novel tool that might be of interest to alphavirus researchers. The authors should address the following concerns: 1. Figure 1A is a little confusing. Please include the schematics for WT CHIKV genome organization alongside with the VRP; also clearly indicate 5'UTR, 3'UTR, and sgRNA promoters in the schematics. 2. The authors should assess the stability of GFP-Luc reporter genes. In Fig 3B-c: very few cells are expressing GFP as compared to nsp1, which raises concerns about the stability of dual reporter transgene in this VRP system. 3. In Figure 4: The authors only show the viral titers in their assessment of continuous VRP production. Do the mosquito cell lines remain viable during the entire 14-day testing period? How can you differentiate between initially produced VRP remaining stable in the supernatant vs newly produced? 4. Panel numbering is confusing for some figures as the authors use A, B, C for main panels and a,b and c for sub-panels. This needs to be rectified. 5.  Staff Comments:

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Thank you for submitting your paper to Microbiology Spectrum. Reviewer 1. Figure 1A is a little confusing. Please include the schematics for WT CHIKV genome organization alongside with the VRP; also clearly indicate 5'UTR, 3'UTR, and sgRNA promoters in the schematics. Response: Thanks for your good suggestion. WT CHIKV genome organization alongside with the VRP; also clearly indicates 5'UTR, 3'UTR, and sgRNA promoters have been included in the schematics of revised Figure 1A. 2. The authors should assess the stability of GFP-Luc reporter genes. In Fig 3B-c: very few cells are expressing GFP as compared to nsp1, which raises concerns about the stability of dual reporter transgene in this VRP system.
Response: We thank the reviewer for pointing out the non-specific issue. The first antibody (rabbit anti-CHIKV nsp1 serum) for CHIKV nsp1 staining is a polyclonal antibody with background signal in IFA. Therefore, false positive cells were observed in nsp1 staining. To avoid non-specific interference, revised Fig 3B has included white arrows to specifically indicate double-positive cells for IFA. 3. In Figure 4: The authors only show the viral titers in their assessment of continuous VRP production. Do the mosquito cell lines remain viable during the entire 14-day testing period? How can you differentiate between initially produced VRP remaining stable in the supernatant vs newly produced? Response: We thank the reviewer for important issues.
Viabilities of both transduced mosquito cells (C6/36 and AP-61) at an MOI of 20 were declined at 5 dpt. As much lower viability of transduced C6/36 cells comparing the viability of transduced AP-61 cells was observed. Rare mosquito cells keep attachment on flask at 8 dpt. However, comparable VRP titers were harvested during dpt 9-14. We recently found equal VRP infectivity on both Vero and AP-61 cells (data not shown), which indicates that initially produced VRP could infect mosquito cells. Thus, the initially produced VRP could be unstable in the supernatant vs the newly produced VRP. Shortening interval time to prevent VRPs infect mosquito cells could improve VRP yield. 4. Panel numbering is confusing for some figures as the authors use A, B, C for main panels and a, b and c for sub-panels. This needs to be rectified.
Response: We thank the reviewer for the valuable suggestion. Labels (a, b and c) for sub-panels of Figure   I am glad to inform you that your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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